DESCRIPTION This proposal seeks to define elements that control the transcriptional regulation of an important virulence factor of a prevalent neonatal human pathogen, Streptococcus agalactiae. The capsular polysaccharide (CPS) or GBS is an important virulence factor and its regulation may contribute to the virulence of this organism. GBS demonstrate growth phase regulation of CPS biosynthesis; however, it is not known what mechanisms control this regulation. The sequence of the CPS biosynthetic operon (cpsIII) suggests that trans-acting factors and cis-acting elements regulate expression of CPS. In this proposal, a genetic approach will be taken to determine if two putative regulatory proteins play a role in the transcriptional regulation of CPS biosynthesis. The cpsY gene (an ORF divergently transcribed from the cpsIII operon), which shows similarity to LysR-type DNA-binding transcriptional regulators, will be inactivated by the insertion of a kanamyin resistance cassette. The cpsX gene is the first gene in the cpsIII operon and encodes a putative transmembrane regulator. The cpsX gene will be inactivated with an in frame deletion. The resulting mutants will be evaluated for transcriptional activity and growth phase regulation of cpsIII transcription. Additionally, regions of symmetry that may reflect cis-acting regulatory elements will be examined for their effects on transcriptional activity of the cpsIII operon.